The long-term objectives of the proposed research are to gain a better understanding of the relationship between the structure and the catalytic function of the cytochrome P450 enzymes and to elucidate the structural basis for the substrate specificities of human cytochromes P450 2B6 and 2E1. The P450 enzymes play critical roles in drug and xenobiotic elimination, drug interactions, carcinogenicity and toxicity, and the biogenesis of sterols and lipids. Major gaps exist in our knowledge of the three-dimensional structures of the active sites of the human P450s in their functional states, the relationship between the three-dimensional structures and the catalytic functions of these enzymes, and the roles that specific residues in the P450s play in the substrate specificity and determination of the metabolic routes preferred for given substrates. The central hypothesis is that specific interactions between residues in and around the active sites of human 2B6 and 2E1 dictate the substrate specificity and the metabolic routes that will be preferred. To test this hypothesis, the Specific Aims of this research proposal are: 1. To identify amino acid residues in human P450s 2E1 and 2B6 whose modification leads to the inactivation of the proteins;2. To investigate the roles of specific amino acid residues in the catalytic function of P450s 2B6 and 2E1;3. To determine the specific step(s) in the P450 catalytic cycle that may be affected by modification of critical amino acid residues in the P450;and 4. To investigate the topologies of the active sites of P450s 2B6 and 2E1 and the effects of modification of selected amino acid residues on the topologies of the active sites. By using the studies with mechanism-based inactivators, site-specific mutagenesis studies, mechanistic studies, and studies on the topologies of the active sites of these two P450s in conjunction with molecular modeling and X-ray crystal structures, we will gain valuable information on the roles of specific amino acid residues in these P450s in their catalytic function. This information will serve as a foundation for understanding and predicting metabolic routes for drugs and carcinogens. The results of these studies will prove to be extremely valuable for developing approaches for selectively modulating the catalytic activity of these enzymes and should provide information that will be extremely valuable in developing methods to decrease the risk of developing cancer and in the design of modalities for the treatment of human cancer. The human cytochromes P450 play a critical role in the metabolism of chemical carcinogens, cancer chemotherapeutic agents, environmental toxins and many drugs. The results of the studies described here should provide us with a better understanding of how the human cytochromes P450 metabolize chemicals that cause cancer and how those chemicals may either be inactivated or converted to forms that are ultimately responsible for causing cancer in people. These studies could ultimately lead to methods to selectively modulate carcinogen metabolism in humans through the development of clinically relevant inhibitors so that metabolic activation is minimized whereas detoxication reactions are enhanced, thereby protecting people from developing cancer as a result of exposure to chemicals that cause cancer.